Rapid chemical signaling between neurons and target cells is dependent upon the precise control of the magnitude and duration of action of neurotransmitters in synaptic spaces. Two principal mechanisms are responsible for rapid transmitter inactivation. Either the neurotransmitter can be enzymatically metabolized to an inactive product, as with the hydrolysis of acetylcholine by acetylcholinesterase, or the neurotransmitter can be actively transported back into presynaptic nerve terminals or surrounding glial cells by one of a large number of specific, pharmacologically distinguishable membrane transport proteins, as reviewed by Snyder, 1970 Biol. Psychiatry 2, 367-389.
Presynaptic nerve endings are enriched for transporters specific to the neurotransmitter they release, thus ensuring constant high levels of neurotransmitters in the nerve terminals, as well as low concentrations in synaptic spaces.
Active transport of neurotransmitters across plasma membranes is energetically coupled to the transmembrane Na.sup.+ gradient generated by (Na.sup.30,K.sup.+)ATPase. Additional ions, including intracellular K.sup.+ and extracellular Cl.sup.-, are also required for transport of many neurotransmitters. Ion sensitivities of neurotransmitter transporters appear to reflect their cotransport with the neurotransmitter during each translocation cycle. These energetic properties, along with clear pharmacological differences, differentiate the Na.sup.+ -dependent plasma membrane transporters from the intracellular, (H.sup.+)ATPase-coupled vesicular transporters that concentrate neurotransmitters in synaptic vesicles for exocytosis. Although glial cells express Na.sup.+ -dependent neurotransmitter transporters, as reported by Schousboe, A. (1981) Int. Rev. Neurobiol. 22, 1045, the quantitative contribution of glial carriers to synaptic transmission is poorly understood. Na.sup.+ -dependent transport processes also mediate the presynaptic accumulation of certain substrates for neurotransmitter synthesis. For example, the rate-limiting step in the biosynthesis of acetylcholine appears to be Na.sup.+ -dependent choline uptake into cholinergic nerve terminals (Kuhar and Murrin, (1978) J. Neurochem. 30, 15-21). It has been proposed that during depolarization, physiologically relevant Ca.sup.2+ -independent release of neurotransmitters may occur by reversal of the Na.sup.+ -dependent uptake process (Schwartz, E. A. (1987) Science 238, 350-355).
High affinity, Na.sup.+ -dependent uptake activities, analogous to the noradrenergic carrier first described at peripheral synapses, have been identified in the mammalian central nervous system (CNS) nerve terminals for the biogenic amine neurotransmitters, including norepinephrine (NE), dopamine (DA), and serotonin (5HT), as reviewed by Snyder, S. H. (1991) Nature 354, 187. The association of high affinity, Na.sup.+ -dependent transport mechanisms in specific neural pathways in the mammalian CNS has provided important information toward the identification of amino acid neurotransmitter candidates. Thus, high affinity, Na.sup.+ -dependent uptake activities have been identified in synaptosomes and brain slices for the excitatory amino acids L-glutamate and L-aspartate and the inhibitory amino acids gammaaminobutyric acid (GABA) and glycine. Presumably, these uptake activities contribute to the regulation of synaptic levels of the transmitter amino acids.
High affinity, Na.sup.+ -dependent uptake of L-proline has also been described in rat brain synaptosomes and slices, as reported by Bennett, et al., (1972) Science 178, 997-999, Peterson and Raghupathy, (1972) J. Neurochem. 19, 1423-1438, Balcar, et al., (1976) Brain Res. 102, 143-151, Hauptman, et al., (1983) FEBS Lett. 161, 301-305, and Nadler, (1987) Nature. 260, 538-540. Furthermore, like the well-established neurotransmitter amino acids, exogenously loaded radiolabeled L-proline is released from brain slices and synaptosomes in a Ca.sup.2+ -dependent manner following K.sup.+ -induced depolarization, as reported by Bennett et al., (1974) Life Sci. 75, 1045-1056; Balcar et al., (1976) Brain Res. 102, 143-151; and Nickolson, (1982) J. Neurochem. 38, 289-292.
In contrast, numerous other amino acids that are not thought to have neurotransmitter roles lack high affinity, Na.sup.+ -dependent synaptosomal uptake activities and are not released to a significant extent from brain slices by K.sup.+ -induced depolarization (Bennett et al., 1974).
The recent cloning and molecular characterization of specific Na.sup.+ -dependent membrane transport proteins for GABA and NE established the presence of a distinct gene family of neurotransmitter transport proteins. These transporters possess significant, greater than 46%, but dispersed amino acid sequence identities and exhibit similar inferred topographies. Both transporters are composed of polypeptides of approximately 600 amino acids and contain approximately 12 hydrophobic stretches of 18-25 amino acids that are thought to form transmembrane domains, analogous to findings with other membrane transport proteins. Amino acid sequence conservation among pharmacologically distinct neurotransmitter transporters likely reflects the involvement of these regions in common transport functions, such as the maintenance of transporter topology and/or the coupling of substrate translocation to the transmembrane Na.sup.+ gradient. However, no significant sequence similarity is observed with other membrane transport proteins, including the mammalian facilitated glucose carriers, the mammalian Na.sup.30 /glucose cotransporter, the prokaryotic Na.sup.+ -dependent cotransporters, and the ATP-binding cassette membrane transporters, including the multidrug resistance P glycoproteins and the cystic fibrosis transmembrane conductance regulator.
Recently, cDNA clones have been identified that encode rat brain DA by Giros et al., (1991) FEBS Lett. 295, 149-154; Kilty, et al., (1991) Science 254, 78-79; and Shimada et al., (1991) Science 254, 576-578, and 5HT by Blakely et al., (1991) Nature 354, 66-70; Hoffman et al., (1991) Science 254, 579-580, transporters. These sequences facilitate further study of the transporters they encode and have potential as diagnostic agents.
It is therefore an object of the present invention to provide a nucleic acid sequence encoding a high affinity, Na.sup.+ -dependent rat brain L-proline transporter.
It is a further object of the present invention to provide probes for related transporter molecules and for studying function and disorders involving these transporter molecules.